The term “autoimmune disease” refers to a group of diseases wherein the immune system mistakenly attacks cells, tissues and organs of a person's own body. Typically, autoimmune diseases involve antibody binding of the body's own components, such as common proteins and lipids. Antibodies that bind to self-compounds (or, more typically, to compounds that are so common that they are found in every organism) are referred to as autoantibodies. As an example, autoantibody binding of phospholipids and/or phospholipid-binding plasma proteins is associated with diseases such as systemic lupus erythematosus (SLE), deep vein and recurrent arterial thrombosis, pulmonary embolisms, recurrent spontaneous abortion, thrombocytopenia, chorea, epilepsy, livedo, idiopathic pulmonary hypertension, rheumatological conditions and a host of collagenous diseases. Other diseases associated with autoantibodies include multiple sclerosis, Crohn's disease, discoid lupus erythematosus, Hashimoto's thyroiditis, psoriasis, diabetes and rheumatoid arthritis. There are about 80 different autoimmune diseases, and as a group, these diseases affect millions of people.
A conventional theory regarding the etiology of autoimmune diseases has been that these diseases are caused by an overproduction of autoantibodies in the diseased individual, possibly due to an overexpression of a gene encoding such autoantibodies. According to this theory, the blood of an affected individual contains an elevated level of the particular autoantibody causing the disease, while the blood of a normal individual contains none of the autoantibody or only a trivial amount. This theory is seemingly supported by conventional assays, in which abundant autoantibodies can be detected in blood, or blood products such as plasma or serum, from subjects having an autoimmune disease, whereas only a zero or minimal amount of autoantibodies can be detected in blood or blood products from subjects that do not have an autoimmune disease.
The present invention is based on the remarkable discovery, reported herein, that blood from normal individuals in fact contains a significant number of autoantibodies, in a wide variety of types and specificities. It is possible to detect and isolate these autoantibodies from blood or a blood product of a normal individual if the blood or blood product is treated by oxidation, by, for example with an oxidizing agent or electric current, according to a method described herein. This discovery of autoantibodies in significant quantities in normal blood is previously unreported and, to the best of the inventor's knowledge, the existence of such autoantibodies in significant quantities in normal blood was completely unknown prior to the present invention.
The discovery of autoantibodies in significant quantities in normal individuals raises the question of why the autoantibodies are not detected in a standard assay (typically based on the binding of an antibody to its corresponding antigen) and why the autoantibodies do not cause disease symptoms in normal individuals.
In experiments described herein, it is shown that by the method of the present invention, autoantibodies can be obtained from biological fluids such as blood from a normal subject by exposing the biological fluid to an oxidizing agent or to a DC electric current, and that the process is reversible. Further, it was found that autoantibodies could be obtained by treating commercial IvIg products. Based on these experiments, a theory for how normal blood could contain autoantibodies without such antibodies being detected through ordinary screening procedures and without such antibodies causing disease is that the autoantibodies freely circulate along with other antibodies but that the antigen binding site of autoantibodies is somehow masked or inactivated in normal individuals. Because of the great quantity and variety of masked antibodies that are discovered according to the present invention, it is evident that such masked antibodies must play a role in healthy individuals. A beneficial role in such masked antibodies is supported by the finding of masked antibodies in human breast milk. Moreover, under the theory that the binding site of autoantibodies can be unmasked by a change in the redox state, it can be theorized that autoimmune diseases are triggered or aggravated by oxidation that unmasks the antigen-binding site of autoantibodies. Moreover, the discovery of masked autoantibodies in cerebral spinal fluid suggests that autoantibodies may be involved in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases, which may be triggered or aggravated by unmasking of autoantibodies. It is known that nitrosylation of proteins in the CNS is one of the earliest measures of Alzheimer's onset. If this is going on in the brain, then this could also unleash the redox reactive autoantibodies to aPL. These aPL antibodies could in turn begin to interact with phospholipids in the brain and cause many of the lesions and shrinkage of the brain which is seen in MRI studies of the Alzheimer patient brains. Further, this theory suggests a more general mechanism by which the binding specificity of certain plasma proteins may be altered.
An immediate practical use of the discovery that forms the basis of the present invention is that it allows for an almost unlimited supply of the newly discovered autoantibodies to be obtained, which autoantibodies can be used as standards in diagnostic kits for the laboratory diagnosis of autoimmune and other aPL-related diseases. Previously, collection of large amounts of autoantibodies for commercial use has been difficult because it was thought that the autoantibodies had to be obtained from individuals having an autoimmune disease or testing positive for autoantibodies in standard assays. The amount of such blood that can be obtained from phlebotomy of individual patients or by pooling blood from a group of patients known to test positive for autoantibodies is limited. Other methods of obtaining autoantibodies, such as screening phage libraries as described in U.S. Pat. No. 5,885,793, may be difficult and time-consuming.
Testing blood samples for the presence or absence of masked antibodies may have important diagnostic value as it might presage or predict what antibodies could appear subsequent to oxidative stress in particular individuals